The goal of this project is to develop readily translatable surrogate biomarkers for hormonalty non-responsive breast cancer chemoprevention trials. For this purpose, surrogate biomarkers will be developed, consisting of breast cell (pre-neoplastic, early neoplastic) secreted proteins that can be assayed for in sera or breast nipple aspirate fluids. These secreted protein biomarkers will be identified and validated in a rat retroviral vector Neu-induced mammary cancer model. Two configurations of this model will be used including one in which ovariectomized rats are given test drugs and the second in which intact rats are treated with tamoxifen and the test drug(s). Drugs to be used include: the monoterpene Perillyl alcohol; the retinoids 9-cis retinoic acid (or Targretin) and 4-HPR; the COX-2 inhibitor Celecoxib; and an EGFR kinase inhibitor. Several of these compounds have been shown to prevent hormonally non-responsive cancer in the rat model proposed. DCIS and carcinoma mammary tissues will be collected from drug-treated and control rats. Differences in gene expression between these tissues will be determined using rat gene expression DNA-chip arrays and rigorous statistical analysis. These differentially expressed genes will be informatically filtered to yield those (approximately 10%) that encode for secreted proteins. Following confirmation of differential gene expression for these selected genes using quantitative-PCR they will be subjected to a proteomic analysis. This analysis will determine if these candidate gene-encoded proteins are differentially expressed in preneoplastic and neoplastic mammary cells of drug treated versus control rats, and whether they are secreted into the blood or mammary ductal fluids in amounts that are modifiable by the drug treatment. Next the human homologues of these rat secreted proteins will be quantified in hormonalty non- responsive breast cancers, breast ductal fluids and sera from patients with various benign, preneoplastic, or neoplastic breast diseases. This project will provide surrogate biomarkers and specific assays for phase II clinical breast cancer prevention trials focused on hormonally non-responsive breast cancer. These surrogate prevention biomarkers, based on proteins that are secreted into blood or breast ductal fluids, will be able to be readily translated into clinical trials in that patient samples for analysis can be collected in a minimally invasive manner.